Men with advanced prostate cancer are treated with hormonal therapy, which leads to an initial response that inevitably recurs in the lethal form of the disease termed castration-resistant prostate cancer (CRPC). While the androgen-signaling axis is the predominant target for therapy in the field, pathways promoting survival and proliferation that are independent of the AR axis need to be identified for potent combinatorial therapeutic strategies. Importantly, therapies aimed at depleting stem/progenitor cell mechanisms, such as self-renewal, have not been adequately explored. We have recently discovered that the stem cell marker Trop2 is a new regulator of self-renewal and proliferation in the prostate and is strongly associated with a castration-resistant state. We have defined a mechanism of action for Trop2 through regulated proteolysis, leading to release of an intracellular domain, similar to activation of Notch. As Trop2 marks and regulates stem cells and is associated with castration-resistance, we propose that blocking Trop2 proteolysis/ activation will inhibit stem-like capacities including self-renewal and proliferation and prevent disease-recurrence. In this proposal, we will utilize clinical specimens, primary regenerated tumors and established cancer xenografts to evaluate Trop2 proteolytic processing as a therapeutic target for future clinical trials in prostate cancer. The goal of AIM 1 is to validate Trop2 as a target in clinical prostate cancer specimens by measuring Trop2, its proteolytic products and downstream effectors in prostate cancer subjects. The goal of AIM 2 is to determine the role of Trop2 in human prostate self-renewal and tumorigenesis, using a dissociated cell tissue recombination strategy to evaluate Trop2+ cells and Trop2 itself in genetically defined primary tumors in vivo. The goal of AIM 3 is to investigate mechanisms to target Trop2 in pre-clinical studies. These experiments will utilize genetic and chemical approaches to establish the role of Trop2 regulated proteolysis, and assess monoclonal antibodies for their ability to interfere with Trop2 processing and tumor growth.